Cancer is a major worldwide public health problem; in the United States alone, approximately 560,000 people died of cancer in 2006. See, e.g., U.S. Mortality Data 2006, National Center for Health Statistics, Centers for Disease Control and Prevention (2010). Many types of cancer have been described in the medical literature. Examples include cancer of blood, bone, skin, lung, colon, breast, prostate, ovary, brain, kidney, bladder, pancreas, and liver, among others. The incidence of cancer continues to climb as the general population ages and as new forms of cancer develop. A continuing need exists for effective therapies to treat subjects with cancer.
Breast cancer is one of the most common types of cancer, especially among women. In the United States, there are about 194,000 new cases of breast cancer and about 40,610 deaths from breast cancer in 2009. See, e.g., Breast Cancer Statistics, National Cancer Institute (2010), available at www.cancer.gov. Among different types of breast cancer, triple negative breast cancer (estrogen receptor (ER)/progesterone receptor/HER-2 negative) is more aggressive than other breast cancer subtypes. No targeted therapy exists for triple negative breast cancer. Triple negative breast cancer has a higher rate of recurrence resulting in death, although the tumors initially appear to respond to chemotherapy. Clearly there is a need to develop effective targeted therapy for triple negative breast cancer.
The Hedgehog (Hh) signaling pathway directs tissue development in embryo, and contributes to tissue homeostasis in adults. Deficient Hh signaling results in defective embryogenesis. Conversely, excessive Hh signaling is associated with an inherited cancer predisposition syndrome (Gorlin Syndrome), and a number of human cancers, including basal cell carcinoma and medulloblastoma. Multiple components of the Hh pathway can be altered in tumors. Studies in tumor cell lines have identified targets that can be exploited for the discovery of human Hh antagonists. Sonic hedgehog, a mammalian version of hedgehog protein, has been shown to stimulate the proliferation of several types of adult stem cells.
The Hh signal is relayed by Patched (Ptc), a 12-transmembrane protein and Smoothened (Smo), a 7-transmembrane protein. Upon binding of the Hh ligand to Ptc, the normal inhibitory effect of Ptc on Smo is relieved, allowing Smo to transduce the Hh signal across the plasma membrane. The signaling cascade initiated by Smo results in activation of Gli transcription factors that translocate into the nucleus where they control transcription of target genes. Smo is the therapeutic target of many drugs designed to treat hedgehog pathway-related diseases including many types of cancers and limb formation abnormalities such as Brachydactyly.
A tight control of the Hh pathway activity is required for proper cellular differentiation and organ formation. Uncontrolled activation of the Hh signaling pathway is associated with malignancies, in particular, those of the brain, skin and muscle, as well as angiogenesis. The Hh pathway has been shown to regulate cell proliferation in adults by the activation of genes involved in cell cycle progression, such as cyclin D, which is involved in the G1-S transition. Sonic hedgehog blocks cell cycle arrest mediated by p21, an inhibitor of cyclin dependent kinases. Hh signaling also induces components in the EGFR pathway (EGF, Her2) involved in proliferation and components in the PDGF (PDGFα) as well as VEGF pathways involved in angiogenesis. Loss-of-function mutations in the Ptc gene have been identified in patients with basal cell nevus syndrome, a hereditary disease characterized by multiple basal cell carcinomas. Dysfunctional Ptc gene mutations have also been associated with a large percentage of sporadic basal cell carcinoma tumors. Loss of Ptc function is thought to cause the uncontrolled Smo signaling in basal cell carcinoma. Activating Smo mutations have been identified in sporadic basal cell carcinoma tumors.
Changes in protein synthesis are directly linked to multiple human cancers. Translation initiation is deregulated in many cancers, including, e.g., lymphoma, breast cancer, head and neck cancer, colorectal cancer, lung cancer, bladder cancer, cervical cancer, and prostate cancer. Many proteins supporting the high rate of cancer cell growth, proliferation, and survival are translated from mRNAs having secondary structures, which have a greater dependence on rate-limiting translation factors such as eukaryotic initiation factor 4E (eIF4E). eIF4E overexpression in tumors can be a predictor for relapse in breast cancer regardless of nodal status and for drug resistance to adjuvant chemotherapy. A high percentage (>60%) of triple negative breast tumors express high levels of eIF4E. The patient group with high levels of eIF4E has a 1.6-fold higher rate of recurrence and a 2.1-fold increase in relative risk for cancer death. High levels of eIF4E drive the cap-dependent translation of proteins responsible for cancer initiation and progression resulting in aggressive phenotypes and enabling the tumors to better survive radiation treatment and chemotherapy. Therefore, it is desirable to regulate protein translation in cancer, in particular, inhibit the rate-limiting steps in protein translation in order to control cell growth and proliferation.
eIF4E, the rate-limiting factor for eukaryotic cap-dependent protein translation, is ubiquitously expressed in multiple breast cancer cell lines. The activity and availability of eIF4E are controlled, e.g., by binding proteins such as 4E-BP1. The activity of these binding proteins is in turn regulated by phosphorylation, particularly by mTOR. eIF4E over-expression along with the concomitant enhanced cap-dependent translation drives cellular transformation and tumorigenesis. eIF4E is a convergence point for many oncogenic pathways and a key factor for malignancy in human cancer tissues and in experimental cancer models. Enhanced translation initiation is found in malignant breast phenotypes. eIF4E over-expression leads to breast carcinoma angiogenesis and progression. eIF4E elevation of 7-fold or more is a strong independent prognostic indicator for breast cancer relapse and death in retrospective and prospective studies. Antisense oligonucleotide therapy down-regulating eIF4E resulted in a reduction of in vivo tumor growth in PC-3 prostate and MDA-MB-231 breast cancer models in mice. No toxicity was observed when 80% knockdown was observed in essential organs, suggesting tumors are more sensitive to cap-dependent translation inhibition than normal tissue.
Cap-dependent translation initiation factor eIF4E and its binding protein 4E-BP1 are major downstream effectors of the PI3K/Akt/mTOR pathway. mTOR and other members of the PI3K/Akt/mTOR family control the establishment and maintenance of cancer phenotypes. The PI3K/Akt/mTOR pathway has been clinically validated as target for cancer therapies. Overactivation of PI3K and Akt is found in a wide range of tumor types. PI3K catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate. This lipid activates Akt protein kinase, which in turn triggers a cascade of responses ranging from cell growth and proliferation to survival and motility. PTEN, a dual specificity phosphatase, is an inhibitor of the PI3K pathway. Second to p53, PTEN is most frequently mutated or deleted in human tumors. Several PI3K inhibitors have been developed in clinical trials. However, due to the integral roles of PI3K and Akt in insulin signaling, it is likely that inhibition of PI3K and Akt activities can lead to abrogated insulin function. Experimental evidence from preclinical models suggests that the blockade of PI3K and Akt results in the loss of insulin signaling in the peripheral tissues and in pancreatic beta cells, potentially leading to hyperglycemia and diabetes. This on-target side effect may limit the therapeutic utility of PI3K and/or Akt inhibitors.
mTOR controls cap-dependent translation through phosphorylation and inactivation of 4E-BP binding protein, thereby activating eIF4E. Activation of eIF4E is required for the translation initiation of mRNAs that have long structured ′5-untranslated regions. Increasing evidence suggests that mTOR, as a central regulator of cell growth and proliferation, controls protein biosynthesis. The mTOR pathway controls translation of mRNAs encoding proteins such as cyclin D1, c-Myc, and ornithine decarboxylase that are essential for G1 cell-cycle progression and S-phase initiation. Inhibition of mTOR results in G1 cell cycle arrest. Rapamycin, an mTOR inhibitor, has significant antitumor activity against many tumor cell lines in the NCI screening as well as in humans. However, formulation, solubility and stability issues have hindered the development of rapamycin. Analogs of rapamycin have been developed to address these issues and have shown promising results in Phase II/III clinical trials. However, preclinical studies and sequential biopsies in patients from a Phase I trial of mTOR inhibitor showed that mTOR inhibition activates Akt via an induced feedback loop. Furthermore, inhibition of mTOR with rapamycin caused exacerbation of diabetes because mTOR serves an important role in insulin signaling.
Therefore, there remains a need for alternative cancer therapeutic agents that are effective and safe, e.g., agents having maximum inhibition of tumor growth, minimal toxicity to normal cells, and minimal on-target side effects in the treated subjects.